| Murine Monocyte and Macrophage Culture
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Author:
Date:
2021-03-20
[Abstract] Myeloid progenitors in the bone marrow generate monocytes, macrophages, granulocytes and most dendritic cells. Even though these innate immune cells are part of the same lineage, each cell type plays a specific and critical role in tissue development, host defense and the generation of adaptive immunity. Protocols have been developed in the past to differentiate myeloid cell types from bone marrow cells, enabling functional investigation and furthering our understanding about their contribution to mammalian physiology. In this protocol, we describe a simple and rapid method to isolate monocytes from murine bone marrow, culture them for up to 5 days and lastly, differentiate them into bone marrow derived macrophages (Figure 1).
Graphic abstract: ...
[摘要] [摘要]骨髓中的骨髓祖细胞产生单核细胞，巨噬细胞，粒细胞和大多数树突状细胞。即使这些先天免疫细胞是同一谱系的一部分，每种细胞类型在组织发育，宿主防御和适应性免疫的产生中也发挥着特定而关键的作用。过去已经开发出区分骨髓细胞和骨髓细胞的协议，以进行功能研究并加深我们对它们对哺乳动物生理学贡献的理解。在该协议中，我们描述了一种简单快速的方法，可从鼠骨髓中分离单核细胞，将其培养长达5天，最后，将它们分化为源自骨髓的巨噬细胞（图1）。
图形摘要：
图1.实验概述，描绘了鼠单核细胞和巨噬细胞培养的步骤
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| Protocol for Isolation, Stimulation and Functional Profiling of Primary and iPSC-derived Human NK Cells
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Author:
Date:
2020-12-05
[Abstract] Natural killer (NK) cells are innate immune cells, characterized by their cytotoxic capacity, and chemokine and cytokine secretion upon activation. Human NK cells are identified by CD56 expression. Circulating NK cells can be further subdivided into the CD56bright (~10%) and CD56dim NK cell subsets (~90%). NK cell-like cells can also be derived from human induced pluripotent stem cells (iPSC). To study the chemokine and cytokine secretion profile of the distinct heterogenous NK cell subsets, intracellular flow cytometry staining can be performed. However, this assay is challenging when the starting material is limited. Alternatively, NK cell subsets can be enriched, sorted, stimulated, and functionally profiled by measuring secreted effector molecules in the supernatant by Luminex. Here, ...
[摘要] [摘要]天然杀伤（NK）细胞是先天性免疫细胞，其特征在于其细胞毒性能力以及活化后的趋化因子和细胞因子分泌。人NK细胞通过CD56表达鉴定。循环的NK细胞可进一步细分为CD56亮（约10％）和CD56暗NK细胞亚群（约90％）。NK细胞样细胞也可以源自人诱导的多能干细胞（iPSC）。为了研究不同的异源NK细胞亚群的趋化因子和细胞因子分泌概况，可以进行细胞内流式细胞仪染色。然而，当起始原料有限时，该测定法具有挑战性。或者，可以通过Luminex测量上清液中分泌的效应子分子来富集，分选，刺激和功能性分析NK细胞亚群。在这里，我们提供了一种快速直接的方案，用于分离和刺激原代NK细胞或iPSC衍生的NK细胞样细胞，并随后检测分泌的细胞因子和趋化因子，这也适用于少量细胞。
[背景]自然杀伤（NK）细胞是先天免疫系统的一部分，提供第一线防御病毒感染和畸形。在人血中，可以基于CD56和CD16表达鉴定出两个不同的NK细胞群体：CD56明亮的CD16 +/-和CD56暗的CD16 + ...
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| Dissecting the Rat Mammary Gland: Isolation, Characterization, and Culture of Purified Mammary Epithelial Cells and Fibroblasts
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Author:
Date:
2020-11-20
[Abstract] With the advent of CRISPR-Cas and the ability to easily modify the genome of diverse organisms, rat models are being increasingly developed to interrogate the genetic events underlying mammary development and tumorigenesis. Protocols for the isolation and characterization of mammary epithelial cell subpopulations have been thoroughly developed for mouse and human tissues, yet there is an increasing need for rat-specific protocols. To date, there are no standard protocols for isolating rat mammary epithelial subpopulations. Analyzing changes in the rat mammary hierarchy will help us elucidate the molecular events in breast cancer, the cells of origin for breast cancer subtypes, and the impact of the tumor microenvironment. Here we describe several methods developed for 1) rat mammary ...
[摘要] [摘要]随着CRISPR-Cas的出现以及能够轻松修饰各种生物的基因组的能力，越来越多地开发大鼠模型来询问乳腺发育和肿瘤发生的遗传事件。已经为小鼠和人类组织彻底开发了用于分离和表征乳腺上皮细胞亚群的方案，但是对大鼠特异性方案的需求却在不断增长。迄今为止，还没有用于分离大鼠乳腺上皮亚群的标准方案。分析大鼠乳腺层次的变化将有助于我们阐明乳腺癌中的分子事件，乳腺癌亚型的起源细胞以及肿瘤微环境的影响。在这里，我们描述为1）大鼠乳腺上皮细胞分离开发的几种方法；2）大鼠乳腺成纤维细胞分离；3）培养大鼠乳腺上皮细胞；通过4）流式细胞仪分析和鉴定大鼠乳腺细胞；5）免疫荧光。源自该协议的细胞可用于多种目的，包括RNAseq ，药物研究，功能测定，基因/蛋白质表达分析和图像分析。
[背景]大多数与乳腺有关的研究都是在小鼠模型和人体样品中进行的。然而，由于其具有类似于人的药代动力学特征和乳腺发育，该疾病的大鼠模型正变得越来越流行（Russo等人，1990；Jiunn等人，2008； Smalley等人，2016）。像人类腺癌一样，大鼠乳腺癌也经历组织学发展阶段（Russo等，1990； Singh等，2000），并且是卵巢激素依赖性的（Thompson等，1998； ...
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